Diphtheria toxin (DT) expression from corynebacteriophage beta is regulated in an iron dependent fashion by the chromosomally encoded diphtheria toxin repressor (DtxR) in C. diphtheriae. Under high iron conditions the DtxR homodimer binds to iron-regulated promoters (IRPs) and represses transcription, thus inhibiting expression of DT and corynebacterial siderophore. Homologs to DtxR have been identified in other Gram-positive bacteria, including Mycobacterium tuberculosis, indicating that DtxR-related repressors may also be involved in iron- dependent regulation of virulence factors in some other Gram positive organisms. Mutational and crystallographic studies showed that there are two metal binding sites in each DtxR monomer. X-ray diffraction data indicate that metal binding site 1 binds both a divalent cation and an anion such as sulfate or phosphate. A model based on these findings is that phosphate may serve as a co-corepressor for DtxR. Preliminary mutagenesis experiments indicate that substitution of an amino acid involved directly in coordination of the anion dramatically decreases the repressor activity of DtxR as measured by expression of beta-galactosidase from a tox operator/promoter-lacZ reporter construct in E. coli. The studies outlined in this proposal are designed to analyze the role of the anion both in metal binding by DtxR and in the signaling pathway for activating DtxR. This will be accomplished by mutagenesis studies of the anion ligands combined with DNA- and metal- binding assays and footprinting analyses with the purified mutant forms of DtxR. X-ray crystallography of the mutant DtxR molecules will be performed in collaboration with Dr. Wim Hol to determine the structural consequences of specific amino acid substitutions that alter DtxR function. These structural, genetic and biochemical analyses will provide new insights into the role of the cation-anion binding site in the activity of DtxR and its homologs as iron-activated global regulators in C. diphtheriae and other pathogens such as M. tuberculosis.